Molecular characterization and expression analysis of Lily-type lectin (SmLTL) in turbot Scophthalmus maximus,and its response to Vibrio anguillarum

A full-length lily-type lectin(Sm LTL) was identified from turbot(Scophthalmus maximus) in this study. By searching database for protein identification and function prediction, Sm LTL were confirmed. The full-length c DNA of Sm LTL is composed of 569 bp and contains a 339 bp ORF that encodes 112 amino acid residues. The Sm LTL peptide is characterized by a specific β-prism architecture and contains three mannose binding sites in a three-fold internal repeat between amino acids 30–99; two of the repeats share the classical mannose binding domain(Qx Dx Nx Vx Y) while the third binding site was similar to other fish-specific binding motifs(Tx Tx Gx Rx V). The primary, secondary, and tertiary structures of Sm LTL were predicted and analyzed, indicating that the S m LTL protein was hydrophilic, contained 5.36% α-helices, 39.29% extended strands, 16.07% β-folds, and 39.29% random coils, and three β-folds. Quantitative realtime polymerase chain reaction(qPCR) analysis revealed that the Sm LTL mRNA was abundantly expressed in skin, gill, and intestine. Low levels of Sm LTL expression were observed in other tissues. The expression of Sm LTL in gill, skin and intestine increased at m RNA level after stimulation of V ibrio anguillarum, our results suggest that Sm LTL serve as the first line of defence against microbial infections and play a pivotal role in the innate mucosal immune system. The current study indicates that Sm LTL is a member of the lilytype lectin family and the information reported here will provide an important foundation for future research on the role of this protein.     

Molecular cloning of the heat shock protein 20 gene from Paphia textile and its expression in response to heat shock

P. textile is an important aquaculture species in China and is mainly distributed in Fujian, Guangdong, and Guangxi Provinces. In this study, an HSP20 c DNA designated Pt HSP20 was cloned from P. textile. The full-length c DNA of Pt HSP20 is 1 090 bp long and contains a 5′ untranslated region(UTR) of 93 bp, a 3′ UTR of 475 bp, and an open reading frame(ORF) of 522 bp. The Pt HSP20 c DNA encodes 173 amino acid residues and has a molecular mass of 20.22 k Da and an isoelectric point of 6.2. Its predicted amino acid sequence shows that Pt HSP20 contains a typical α-crystallin domain(residues 77–171) and three polyadenylation signal-sequences at the C-terminus. According to an amino acid sequence alignment, Pt HSP20 shows moderate homology to other mollusk s HSPs. Pt HSP20 m RNA was present in all of the test tissues including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, with the highest concentration found in the gonad. Under the stress of high temperature, the expression of Pt HSP20 m RNA was down-regulated in all of the tissues except the adductor muscle and gonad.     

Antioxidation and ATPase activity in the gill of mud crab Scylla serrata under cold stress

Mud crab (Scylla serrata) is an important commercial crustacean in China. An experiment was designed to study the effect of cold stress on S. serrata. After a one-week adaptation at 28oC, the temperature is suddenly reduced to 4oC. The crabs were sampled every 2 h for 10 h and dissected immediately to measure the enzyme activity. The crabs at room temperature (28oC) were used as the control group. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), the content of malondialdehyde (MDA) and the activity of 4 ATPases (Na , K -ATPase; Mg2 -ATPase; Ca2 -ATPase; Ca2 , Mg2 -ATPase) were measured biochemically. In contrast to the control group, the SOD activity increased significantly from 2 to 6 h after the cold stress, and then decreased. The CAT and GPX activities increased in 2 h, and then decreased gradually. The content of MDA increased gradually in 4 h. The activity of Na , K -ATPase decreased in 2 h, increased up to the top value at Hour 6, then decreased again. The activities of Mg2 -ATPase, Ca2 -ATPase and Ca2 , Mg2 -ATPase increased significantly in 6 h, insignificantly in any other hours. Under cold stress, the activity of antioxidative enzymes in S. serrata was reduced at first then stabilized, ROS-scavenging weakened, and MDA accumulated gradually in the gill after 6 h. The activity of the 4 ATPases in the crab decreased after 6 h, suggesting that the ability to regulate ion concentration has been paralyzed. Therefore, the maximum period to sustain healthy meat in the crab under cold stress is 6 hours.     

Cloning and stage-specific expression of CK-M1 gene during metamorphosis of Japanese flounder,Paralichthys olivaceus

The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study.     

Characterization of SNAP-25 gene from marine teleostean, Lateolabrax japonicus

The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.     

Effect of Salinity on Hemolymph Osmotic Pressure, Sodium Concentration and Na^＋-K^＋-ATPase Activity of Gill of Chinese Crab, Eriocheir sinensis

The effects of salinity on hemolymph osmotic pressure, Na^＋ concentration and Na^＋-K^＋-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na^＋ concentration increased significantly （P〈0.05）, and the Na^＋-K^＋-ATPase activity of gills decreased significantly （P〈0.05） when salinity increased from 0 to 16. The hemolymph osmotic pressure and Na^＋ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na^＋-K^＋-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na^＋ concentration and lower Na^＋-K^＋-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct （P〉0.05）; However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1d, and then tended to be stable from day 1 to day 15.     

Characterization,Expression and Function Analysis of DAX1 Gene of Scallop （Chlamys farreri Jones and Preston 1904） During Its Gametogenesis

DAX 1,a member of nuclear receptor superfamily,has a function in the sex determination and gonadal differentiation of several vertebrate species.However,little information about DAX1 of invertebrates is available.Here we cloned a homolog of scallop （Chlamys farreri Jones and Preston 1904） dax1,Cf-dax1,and determined its expression characteristics at mRNA and protein levels.The cDNA sequence of Cf-dax1 was 2093 bp in length,including 1404 bp open reading frame （ORF） encoding 467 amino acids.Unlike those of vertebrates,no conserved LXXLL-related motif was found in the putative DNA binding region of Cf-DAX1.Fluorescence in situ hybridization showed that Cf-dax1 located on the short arm of a pair of subtelocentric chromosomes.Tissue distribution analysis using semi-quantitative RT-PCR revealed that Cf-dax1 expressed widely in adult scallop tissues,with the highest expression level found in adductor muscle,moderate level in mantle,gill and testis,and low level in kidney,ovary and hepatopancreas.The result of quantitative real-time PCR indicated that the expression of Cf-dax1 was significantly higher （P＜0.05） in testis than in ovary at the same stage,showing a sex-dimorphic expression pattern.Furthermore,immunohistochemical detection found that Cf-DAX1 mainly located in spermatogonia and spermatocytes of testis and in oogonia and oocytes of ovary,implying that DAX1 may involve in gametogenesis of bivalves.     

A Carboxymethyl Cellulase from a Marine Yeast (Aureobasidium pullulans 98): Its Purification,Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase(CMCase) in the supernatant of the culture of A. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U(mg protein)-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0 k Da. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other fungi. The optimal p H of the enzyme was 5.6, and the activity profile was stable in a range of acidity(p H 5.0–6.0). The enzyme was activated by Na+, Mg2+, Ca2+, K+, Fe2+ and Cu2+, however, it was inhibited by Fe3+, Ba2+, Zn2+, Mn2+ and Ag+. Km and Vmax values of the purified enzyme were 4.7 mg m L-1 and 0.57 μmol L-1 min-1(mg protein)-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose(CMC) after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading frame of 954 bp(EU978473). The protein deduced contained the conserved domain of cellulase superfamily(glucosyl hydrolase family 5). The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.     

Effect of cadmium on the defense response of Pacific oyster Crassostrea gigas to Listonella anguillarum challenge

Heavy metal pollution can affect the immune capability of organisms.We evaluated the effect of cadmium(Cd) on the defense responses of the Pacific oyster Crassostrea gigas to Listonella anguillarum challenge.The activities of several important defensive enzymes,including superoxide dismutase(SOD),glutathione peroxidase(GPx),acid phosphatase(ACP),Na+,K+-ATPase in gills and hepatopancreas,and phenoloxidase-like(POL) enzyme in hemolymph were assayed.In addition,the expression levels of several genes,including heat shock protein 90(HSP90),metallothionein(MT),and bactericidal/permeability increasing(BPI) protein were quantified by fluorescent quantitative PCR.The enzyme activities of SOD,ACP,POL,and GPx in hepatopancreas,and the expression of HSP90 were down-regulated,whereas GPx activity in the gill,Na+,K+-ATPase activities in both tissues,and MT expression was increased in Cdexposed oysters post L.anguillarum challenge.However,BPI expression was not significantly altered by co-stress of L.anguillarum infection and cadmium exposure.Our results suggest that cadmium exposure alters the oysters’ immune responses and energy metabolism following vibrio infection.     

Changes in growth and osmoregulation during acclimation to saltwater in juvenile Amur sturgeon Acipenser schrenckii

We evaluated the ability of juvenile Amur sturgeon (Acipenser schrenckii) to osmoregulate and grow in saltwater. Hatchery-reared juveniles (mean weight 106.8 g, 5-month old) were transferred from freshwater to 10, 20, and 25 salinity saltwater over a period of 20 d. We measured the growth, serum osmolality, ion concentrations, and Na+/K+-ATPase activity. In addition, we prepared samples of gill tissue to quantify morphological changes in gill ultrastructure. Rearing in up to 25 saltwater for 30 d had no significant effect on growth. Similarly, serum osmolality and ion concentrations were similar to levels reported in other teleosts following acclimation to saltwater. Serum osmolality and Na+, Cl- concentrations increased significantly with the initial increase in salinity. Afterwards, levels tended to stabilize and then decrease. Serum K+ levels did not change during acclimation to saltwater. Gill Na+/K+-ATPase activity increased initially as salinity was increased. However, the activity later decreased and, finally stabilized at 3.7±0.1 μmol Pi/mg·prot·h in 25 saltwater (1.6 times higher than the level in those in freshwater). In fish that were held only in freshwater, the chloride cells were located in the interlamellar regions of the filament and at the base of the lamella. Following acclimation to 25 saltwater for 30 d, the number and size of chloride cells increased significantly. Our results suggest that juvenile Amur sturgeon is able to tolerate, and grow in, relatively high concentrations of saltwater.     

Phospholipid Compositions in Portunus trituberculatus Larvae at Different Developmental Stages

Phospholipids are used to improve the growth and survival of Portunus trituberculatus,a widely cultured crab species in China.However,only total phospholipids or several classes are applied in crab diets.In this study,we employed a targeted lipidomic method to investigate the comprehensive phospholipid composition in P.trituberculatus larvae and reveal the changing phospholipid profile over the larval development.Results showed that P.trituberculatus larvae contain 112 phospholipid species belonging to 10 phospholipid classes,in which phosphatidylcholine(PC)and phosphatidylethanolamine(PE)species are the most abundant,and PC,PE,phosphatidic acid,and phosphatidylserine(PS)are with high concentrations.The levels of all phospholipids significantly changed with larval development,which was highlighted by the downward parabolic changes in PE,phosphatidylglycerol,phosphatidylino-sitol,PS,lysophosphatidic acid,and sphingomyelin levels.In addition,nearly all phospholipid species were depleted at the M stage,which probably contributed to the mass mortality of crab larvae.These findings on the composition and alterations of phospholipids in P.trituberculatus larvae provide novel perspectives for the targeted supplementation of phospholipids in crab diets.Our work also highlights the use of targeted UHPLC-MS lipidomics in understanding the changes of phospholipids during crab development.     

Air Temperature and Emersion Time Can Affect the Survival Rate and Ammonium Loading of Swimming Crab Portunus trituberculatus Exposed to Air

Ammonium overloading is a common response of aquatic organisms to air exposure during transport. This study elucidated the relationship between ammonium overloading and mortality of crab Portunus trituberculatus. Additionally, we also explored the effects of emersion time and air temperature on ammonium loading and concomitant physiological change. To test the air temperature effect, the crab was exposed to 16, 23 and 30℃ in air for 3 h, respectively, and then recovered in seawater at 23℃ for 12 h. To test the emersion time effect, crab was exposed to 23℃ in air for 0.5 and 3 h, respectively, and then recovered in seawater at 23℃ for 12 h. In the control group, crab was always immersed at 23℃. At each time interval(0.5, 1.5 and 3 h during air exposure and 0.5, 2, 4 and 12 h during recovery), ammonium excretion rate, level of total ammonium, total free amino acids and urea concentration in hemolymph and the hepatopancreas enzyme activity involved in detoxifying ammonium were analysed. Results showed that crab mortality was positively related with emersion time and temperature while ammonium loading was lower at 16 and 30℃ than at 23℃. For crab experiencing thermal inconsistence of culture media(i.e., 16 or 30℃), they were higher in ammonium excretion rate and activities of ammonium detoxification enzymes, which may be the reason that they had a lower ammonium loading. Prolonged emersion time(3.0 h vs. 0.5 h) increased the ammonium overloading and the activity of ammonium detoxification pathways in crab. Our results demonstrated that emersion-induced ammonium overloading may not be the main reason leading to P. trituberculatus death during air exposure and subsequent recovery. When the culture medium changed, thermal variation, compared with constant temperature, could reduce ammonium overloading in crab by elevating the activities of ammonium detoxification enzymes and ammonium excretion rate during recovery period.     

Cloning of the cDNA encoding adenosine 5′-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5’-UTR of 78 bp, a 3’-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.     

Isolation and Characterization of Collagen from Squid(Ommastrephes bartrami) Skin

Collagen of squid(Ommastrephes bartrami) skin was examined in the present study. Histology showed that collagen fiber in the skin was partially cross-linked with muscle fiber. Acid-solubilized collagen(ASC) and pepsin-solubilized collagen(PSC) were extracted from the skin and characterized. The results of amino acid composition and electrophoretic patterns revealed that ASC and PSC were both type Ⅰ collagen,containing α1 and α2 chains. FTIR(fourier transform infrared spectroscopy) investigations confirmed t...